Mycobacterium tuberculosis is an ancient pathogen that continues to flourish within the modern human population, causing 1.9 million deaths annually across the globe. Additionally, M. tuberculosis is a major AIDS- related opportunistic pathogen. The hallmark feature of tuberculosis, the granuloma, is a defined cellular aggregate within the lung tissue, surrounding and containing the initial focus of infection. Granuloma formation has long been understood as the primary host defense against M. tuberculosis; however, this structure also serves as a reservoir for the long-term maintenance of viable mycobacteria within an infected individual, and recent studies indicate that the ability of M. tuberculosis to initiate granuloma development is an important virulence determinant for this organism. Therefore, molecular factors that contribute to formation of the granuloma represent potential targets that could disrupt the ability of M. tuberculosis to efficiently infect and persist within the human population. Recently, our laboratory has characterized an M. tuberculosis-encoded adenylate cyclase (Rv0386) responsible for the production of 3', 5'-cyclic adenosine monophosphate (cAMP) that is directly injected into the host cell, and preliminary data indicate that this mycobacterial-derived cAMP may affect signaling events involved in development of the granuloma. We hypothesize that the active efflux of cAMP from M. tuberculosis influences the host response to promote a local environment favorable for mycobacterial survival within the granuloma structure, and we have designed three specific research aims to address this hypothesis. First, we will examine how M. tuberculosis secretes cAMP across the bacterial membrane into the host cell. The hydrophilic nature of the cAMP molecule precludes its diffusion across biological membranes, and we will utilize an extensive mutant collection to identify M. tuberculosis genes important for cAMP efflux during growth in broth culture. Second, we will evaluate how the mycobacterial-derived cAMP affects host cell signaling events involved in the production and secretion of matrix metalloproteinases (MMPs). These enzymes are responsible for breakdown of the lung extracellular matrix, a process that is critical for granuloma formation. We propose to use the M. tuberculosis adenylate cyclase mutant (JHU-0386), as well as a complemented strain, to analyze the role of cAMP on the gene and protein expression of human MMPs, as well as on the signaling events leading to this expression, following M. tuberculosis infection in cultured human monocytic cells. Third, we will utilize the JHU-0386 mutant to characterize the role of the mycobacterial-derived cAMP during in vivo granuloma formation and development within a novel rabbit model of tuberculosis. Overall, in the endeavor to further understand M. tuberculosis pathogenesis and identify new therapeutic targets against this organism, this project combines in vitro and in vivo experiments to study both bacterial and host cell factors involved in development of the lung granuloma following an M. tuberculosis infection.